Enterovirus-A71 exploits RAB11 to recruit chaperones for virus morphogenesis

Background Enterovirus 71 (EV-A71) causes Hand, Foot and Mouth Disease (HFMD) in children and has been associated with neurological complications. The molecular mechanisms involved in EV-A71 pathogenesis have remained elusive. Methods A siRNA screen in EV-A71 infected-motor neurons was performed targeting 112 genes involved in intracellular membrane trafficking, followed by validation of the top four hits using deconvoluted siRNA. Downstream approaches including viral entry by-pass, intracellular viral genome quantification by qPCR, Western blot analyses, and Luciferase reporter assays allowed determine the stage of the infection cycle the top candidate, RAB11A was involved in. Proximity ligation assay, co-immunoprecipitation and multiplex confocal imaging were employed to study interactions between viral components and RAB11A. Dominant negative and constitutively active RAB11A constructs were used to determine the importance of the protein’s GTPase activity during EV-A71 infection. Mass spectrometry and protein interaction analyses were employed for the identification of RAB11A’s host interacting partners during infection. Results Small GTPase RAB11A was identified as a novel pro-viral host factor during EV-A71 infection. RAB11A and RAB11B isoforms were interchangeably exploited by strains from major EV-A71 genogroups and by Coxsackievirus A16, another major causative agent of HFMD. We showed that RAB11A was not involved in viral entry, IRES-mediated protein translation, viral genome replication, and virus exit. RAB11A co-localized with replication organelles where it interacted with structural and non-structural viral components. Over-expression of dominant negative (S25N; GDP-bound) and constitutively active (Q70L; GTP-bound) RAB11A mutants had no effect on EV-A71 infection outcome, ruling out RAB11A’s involvement in intracellular trafficking of viral or host components. Instead, decreased ratio of intracellular mature viral particles to viral RNA copies and increased VP0:VP2 ratio in siRAB11-treated cells supported a role in provirion maturation hallmarked by VP0 cleavage into VP2 and VP4. Finally, chaperones, not trafficking and transporter proteins, were found to be RAB11A’s top interacting partners during EV-A71 infection. Among which, CCT8 subunit from the chaperone complex TRiC/CCT was further validated and shown to interact with viral structural proteins specifically, representing yet another novel pro-viral host factor during EV-A71 infection. Conclusions This study describes a novel, unconventional role for RAB11A during viral infection where it participates in the complex process of virus morphogenesis by recruiting essential chaperone proteins. Supplementary Information The online version contains supplementary material available at 10.1186/s12929-024-01053-2.

Co-localization and interaction between RAB11A with CCT8 was assessed by proximity ligation assay (PLA).(A) SH-SY5Y cells were infected with EV-A71 S41 (MOI 0.1).At 24 h.p.i., the cells were fixed and permeabilized before staining with anti-RAB11A paired with either anti-CCT8 or IgG isotype control antibodies.The cells were then stained with secondary antibodies conjugated with DNA probes.Ligation and polymerase chain reaction were then carried out for signal amplification.These cells were further stained using in-house conjugated EV-A71 VP1 antibody to identify infected cells.Nuclei were stained with DAPI.Images were captured at 100X magnification under Olympus FV3000 confocal microscope.Images were further zoomed in by 150% using Fiji software.

Figure S1 .
Figure S1.Effects of RAB11A and RAB11B siRNA KD in SH-SY5Y cells.SH-SY5Y cells were transfected with deconvoluted human siRAB11A #10, siRAB11B #9, a mix of both or siNTC, followed by infection with EV-A71 S41 (MOI 0.1) at 48h.p.t.At 24h.p.i, the culture supernatants and cells were harvested.(A) Virus titers in the cultured supernatants were determined by plaque assay.Statistical analysis was performed using Kruskal-Wallis test against siNTC treatment (*p<0.05,**p<0.01,***p<0.001,****p<0.0001).The percentage of viral titer reduction was indicated above the asterisk.(B) The cell lysates were subjected to Western blot analysis using anti-VP2, anti-RAB11A, anti-RAB11B and anti B-actin antibodies.(C) VP0:VP2 ratio relative to NTC, based on signal band intensity measured by ImageLab.(D) AlamarBlue assay was performed on uninfected siRNA-treated cells.The readout was relative to non-siRNA-treated cells to determine the percentage of cell viability.Percentage above 70% (indicated by the dotted line) was considered non-cytotoxic.

Figure S3 :
Figure S3: EV-A71 viral kinetic profile in siRAB11-treated cells.SH-SY5Y cells were transfected with siRAB11 or with siNTC, followed by infection with EV-A71 S41 (MOI 0.1) at 48h.p.t.The culture supernatants and cell lysates were harvested at various timepoints ranging from 6h.p.i to 48h.p.i.(A) Virus titers in the culture supernatants were determined by plaque assay.Statistical analysis was performed using Mann-Whitney U test against siNTC treatment (*p<0.05,**p<0.01,***p<0.001,****p<0.0001).The percentage of virus titer reduction in siRab11-treated samples relative to siNTC samples was indicated above the asterisk.(B) The cell lysates were subjected to Western blot analysis using anti-VP2, anti-3C, anti-RAB11A, anti-RAB11B and anti B-actin primary antibodies.Band intensities were determined using ImageLab and were normalised to Beta-actin.(C) VP0, VP2, 3C and 3CD band intensities of siRAB11 and siNTC treatment were calculated.(D) VP2:VP0 ratio were further computed.(E) AlamarBlue assay was performed on uninfected siRNAtreated cells at 48 hours post-transfection.The readout for each treatment was relative to untreated cells to determine the percentage of cell viability.Percentage above 70% (indicated by the dotted line) was considered non-cytotoxic.

Figure S5 :
Figure S5: Effect of guanidine hydrochloride on Luciferase activity in SH-SY5Y cells.SH-SY5Y cells were treated with siRab11 or siNTC, then transfected with 500ng of in vitro transcribed EV71-Luc RNA.At 4 hours posttransfection, culture medium was replaced with complete growth medium containing 2mM of GuHCl or 0.05% DMSO.(A) Luciferase activity and (D) cytotoxicity were measured at 24 hours post-transfection.(B) The ratio of luciferase signal in GuHCl-treated samples relative to that in samples treated with 0.05% DMSO was determined.(C) siRab11-treated cells were also harvested to assess the siRAB11 KD efficiency by Western blot, using anti-RAB11A, anti-RAB11B and Anti-B-actin primary antibodies.Statistical analysis was performed for (A) and (B) using Mann-Whitney U test against siNTC treatment (*p<0.05,**p<0.01,***p<0.001,****p<0.0001).

Figure S6 .
Figure S6.Interactions between RAB11A and viral protein 3C/3CD in various subcellular compartments over time.SH-SY5Y cells were infected with EV-A71 S41 (MOI 0.1).At the indicated time points post-infection, the cells were fixed and permeabilized, followed by staining with anti-RAB11A and anti-3C primary antibodies, and labelled secondary antibodies.Cells were then further stained with antibodies specific to compartment markers calreticulin for endoplasmic reticulum (A), GM130 for Golgi apparatus (B) and transferrin receptor (Tfr) for small recycling endosomes (C) prior to DAPI staining.Confocal images were captured under 100X objective and were analyzed using Fiji software to determine the voxels/pixels that represent 3 channel (green, red, magenta) colocalization.Briefly, a mask representing the co-localization of RAB11A and each viral component was delineated and subsequently overlayed with compartment marker signals to generate the 'colocalized voxels' images shown on the far right column.(D) Pearson correlation coefficient (PCC) values were computed using Fiji software.PCC value of 0 = no co-localization; 0.1 -0.3 = weak co-localization; 0.3 -0.5 = moderate co-localization; 0.5-1 = strong co-localization.Statistical analysis was performed using Kruskal-Wallis test with Dunn correction against siNTC treatment (*p<0.05,**p<0.01,***p<0.001,****p<0.0001).

Figure S7 .
Figure S7.Interactions between RAB11A and viral proteins VP1, VP2, 3D, 3C, or dsRNA in various subcellular compartments at 6 hours post-infection.SH-SY5Y cells were infected with EV-A71 S41 (MOI 0.1).At the indicated time points post-infection, the cells were fixed and permeabilized, followed by staining with anti-RAB11A and anti-VP2, VP1, 3C, 3D or dsRNA primary antibodies, and labelled secondary antibodies.Cells were then further stained with antibodies specific to compartment markers calreticulin for endoplasmic reticulum (A), GM130 for Golgi apparatus (B) and transferrin receptor (Tfr) for small recycling endosomes (C) prior to DAPI staining.Confocal images were captured under 100X objective and were analyzed using Fiji software to determine the voxels/pixels that represent three channels (green, red, magenta) co-localization.Briefly, a mask representing the co-localization of RAB11A and each viral component was delineated and subsequently overlayed with compartment marker signals to generate the 'co-localized voxels' images shown on the far right column.(D) Pearson correlation coefficient (PCC) values were computed using Fiji software.PCC value of 0 = no co-localization; 0.1 -0.3 = weak co-localization; 0.3 -0.5 = moderate co-localization; 0.5-1 = strong co-localization.Statistical analysis was performed using Kruskal-Wallis test with Dunn correction against siNTC treatment (*p<0.05,**p<0.01,***p<0.001,****p<0.0001).

Figure S8 .
Figure S8.Distribution patterns of overexpressed RAB11A WT, DN (RAB11A S25N) and CA (RAB11A Q70L) in infected and uninfected SH-SY5Y cells.Images taken from Fig. S8 panel B were further zoomed in (200%) using Fiji software.White arrows indicate specific distribution patterns that were lost in infected cells.

Figure S10 .
Figure S10.Total protein content and RAB11A expression in input samples prior to Co-IP for mass spectrometry (Fig. 9).10ug of each input sample was used for gel electrophoresis.(A&B) Western blot analysis using anti-RAB11A, anti-VP2 and anti-B-actin.(C) TGX stain-free gel to visualize the total protein content in each sample.

Figure S11 .
Figure S11.RAB11A and CCT8 co-localization and interaction.Co-localization and interaction between RAB11A with CCT8 was assessed by proximity ligation assay (PLA).(A) SH-SY5Y cells were infected with EV-A71 S41 (MOI 0.1).At 24 h.p.i., the cells were fixed and permeabilized before staining with anti-RAB11A paired with either anti-CCT8 or IgG isotype control antibodies.The cells were then stained with secondary antibodies conjugated with DNA probes.Ligation and polymerase chain reaction were then carried out for signal amplification.These cells were further stained using in-house conjugated EV-A71 VP1 antibody to identify infected cells.Nuclei were stained with DAPI.Images were captured at 100X magnification under Olympus FV3000 confocal microscope.Images were further zoomed in by 150% using Fiji software.